Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.1.34 (lipoprotein lipase)
7,025 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rat platelets secrete two types of phospholipases upon stimulation; one is type II phospholipase A2 and the other is serine-phospholipid-selective phospholipase A. In the current study we purified serine-phospholipid-selective phospholipase A and cloned its cDNA. The final preparation, purified from extracellular medium of activated rat platelets, gave a 55-kDa protein band on SDS-polyacrylamide gel electrophoresis. [3H]Diisopropyl fluorophosphate, an inhibitor of the enzyme, labeled the 55-kDa protein, suggesting that this polypeptide possesses active serine residues. The cDNA for the enzyme was cloned from a rat megakaryocyte cDNA library. The predicted 456-amino acid sequence contains a putative short N-terminal signal sequence and a GXSXG sequence, which is a motif of an active serine residue of serine esterase. Amino acid sequence homology analysis revealed that the enzyme shares about 30% homology with mammalian lipases (lipoprotein lipase, hepatic lipase, and pancreatic lipase). Regions surrounding the putative active serine, histidine, and aspartic acid, which may form a "lipase triad," were highly conserved among these enzymes. The recombinant protein, which we expressed in Sf9 insect cells using the baculovirus system, hydrolyzed a fatty acyl residue at the sn-1 position of lysophosphatidylserine and phosphatidylserine, but did not appreciably hydrolyze phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, and triglyceride. The present enzyme, named phosphatidylserine-phospholipase A1, is the first phospholipase that exclusively hydrolyses the sn-1 position and has a strict head group specificity for the substrate.
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PMID:Serine phospholipid-specific phospholipase A that is secreted from activated platelets. A new member of the lipase family. 899 22

This study was performed to evaluate the effect of human recombinant basic fibroblast growth factor on arachidonic acid release from rat pancreatic acini and to determine the cellular mechanism involved. From enzymatic assays, basic fibroblast growth factor did not significantly stimulate phospholipase A2 activity, whereas it significantly increased diacylglycerol lipase activity. Validity of phospholipase A2 or diacylglycerol lipase inhibitors was confirmed by their ability to inhibit phospholipase A2 or diacylglycerol lipase activities. Basic fibroblast growth factor increased intracellular accumulation and extracellular release of arachidonic acid from metabolically labelled acinar cells in a concentration- and time-dependent manner. This effect was maximal with 50 pM basic fibroblast growth factor and became significant after a 5-min incubation period. The protein tyrosine kinase inhibitor, 0.5 mM genistein, inhibited arachidonic acid release in basic fibroblast growth factor-stimulated acini, whereas 100 microM vanadate, a protein tyrosine phosphatase inhibitor, enhanced arachidonic acid release. Two phospholipase A2 inhibitors, mepacrine and aristolochic acid, failed to attenuate basic fibroblast growth factor-stimulated arachidonic acid release. A diacylglycerol lipase inhibitor RHC 80267 at 150 microM and 50 microM completely inhibited 50 pM basic fibroblast growth factor-induced intracellular accumulation and extracellular release of arachidonic acid, respectively. Furthermore, basic fibroblast growth factor stimulated arachidonic acid release was also inhibited by 10 microM U73122 and by 100 nM staurosporine, phospholipase C and protein kinase C respective inhibitors. Wortmannin, an inhibitor of basic fibroblast growth factor-stimulated phospholipase D, did not affect arachidonic acid release. 100 nM 4 beta-phorbol 12-myristate 13-acetate also increased arachidonic acid release, an effect also inhibited by staurosporine. Taken together, these data demonstrate activation of diacylglycerol lipase and arachidonic acid release in pancreatic acini upon stimulation by basic fibroblast growth factor, and strongly indicate that arachidonic acid release in response to basic fibroblast growth factor depends upon the sequential action of tyrosine kinase, phospholipase C, protein kinase C and diacylglycerol lipase but not from phospholipase A2 not phospholipase D activation.
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PMID:Basic fibroblast growth factor-stimulated arachidonic acid release in rat pancreatic acini: sequential action of tyrosine kinase, phospholipase C, protein kinase C and diacylglycerol lipase. 902 13

The breakdown of normal substrates by lipases requires an interfacial binding step prior to hydrolysis. Interfacial binding and subsequent hydrolysis will be affected by the lipid components and hence physical properties of the substrate surface. In order to investigate in detail the effect of lipid structure on the activity of lipoprotein lipase (LPL), triolein-containing emulsion particles of defined composition have been used as substrates. In addition, lipase activity has been measured using a continuous fluorescence displacement assay that monitors the release of long-chain fatty acids as an alternative to normal radiochemical assays. Using this fluorescence assay, rates of hydrolysis of triolein were the same as when using a standard radiochemical assay under identical conditions. Activation by apolipoprotein CII was very similar by both methods; however, the extent of activation (2-3-fold) was less than has been reported previously using different assay conditions. In order to investigate the effect of cholesterol on LPL activity, emulsion particles were prepared in which the cholesterol/egg-phosphatidylcholine ratio was increased up to a 1:1 molar ratio. A pronounced stimulatory effect of cholesterol was observed under these assay conditions, with up to a 5-fold increase in rate compared with emulsion particles without cholesterol. Since high molar ratios of cholesterol are reported to exclude triacylglycerol from the phospholipid surface [Spooner and Small (1987) Biochemistry 26, 5820-5825], these results are not consistent with a mechanism involving LPL hydrolysis of surface triacylglycerol. Instead, they support an interfacial penetration model, allowing the enzyme's active site direct access to triacylglycerol in the lipoprotein core. Perturbation of the surface phospholipid monolayer of the emulsion particle as a result of hydrolysis by Naja naja phospholipase A2 resulted in a 10-fold activation of LPL, providing further support for an interfacial penetration model. The stimulatory effect of apolipoprotein CII was not modulated by modification of the interface with cholesterol.
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PMID:Effect of lipid composition on lipoprotein lipase activity measured by a continuous fluorescence assay: effect of cholesterol supports an interfacial surface penetration model. 903 72

In rat inner medullary collecting duct (IMCD) cells in primary culture, hypotonic stress induces Ca2+ transients consisting of an early peak phase caused by a Ca2+ release from intracellular stores and a subsequent plateau phase that involves Ca2+ entry from the extracellular milieu. In the present study, the mechanisms by which cell swelling is transduced into the Ca2+ release were investigated. The free intracellular Ca2+ concentration ([Ca2+]i) was measured using the fluorescent dye fura-2 and cell volume using a confocal laser scanning microscope. In control experiments, after reduction of extracellular osmolarity from 600 to 300 mosmol/l, by omission of sucrose, [Ca2+]i rapidly increased from 106 +/- 9 nmol/l to a peak value of 405 +/- 22 nmol/l (P </= 0.05) and thereafter reached a steady-state of 230 +/- 23 nmol/l. In low-Ca2+ conditions (10 nmol/l), the reduction of osmolarity evoked only a transient increase of [Ca2+]i by 182 +/- 11 nmol/l (P </= 0.05), which reflected Ca2+ release from intracellular stores. Hyposmotic stress had no effect on inositol 1,4,5-triphosphate (IP3) production measured by a [3H]IP3 radioreceptor assay. Preincubation with 100 micromol/l ETYA (a non-metabolisible derivative of arachidonic acid) reduced the Ca2+ response to hyposmotic stress under high and low Ca2+ conditions (87 and 85% inhibition respectively) as well as the regulatory volume decrease (RVD). Extracellular application of arachidonic acid in isotonic medium led to an increase in [Ca2+]i under high and low Ca2+ conditions. Pretreatment of IMCD cells with 50 microg/ml D609 (a phosphatidylcholine-directed phospholipase C inhibitor) or with 200 micromol/l propranolol (a phosphatidate phosphohydrolase inhibitor) reduced the hypotonic Ca2+ response more strongly than pretreatment with 5 micromol/l BPhB (a phospholipase A2 inhibitor). The Ca2+ response was also suppressed after preincubation with 200 micromol/l RHC 80267 (a diacylglycerol lipase inhibitor). Preincubation with 50 ng/ml pertussis toxin (a G-protein inhibitor) reduced the transient component of the Ca2+ response partially. We conclude that G-proteins, phosphatidylcholine-directed phospholipase C, phospholipase A2, diacylglycerol lipase and arachidonic acid, but not IP3, are involved in the mechanisms by which Ca2+ is released from the intracellular stores during RVD in IMCD cells.
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PMID:Arachidonic acid as a second messenger for hypotonicity-induced calcium transients in rat IMCD cells. 906 39

We investigated the effect of extracellular ATP on phosphatidylcholine-hydrolyzing phospholipase D activity and the role of phospholipase D activation in extracellular ATP-induced arachidonic acid release in cultured rat aortic smooth muscle cells. ATP significantly stimulated the formation of choline in a dose-dependent manner in the range between 0.01 and 0.5 mmol/L. However, ATP had no effect on the formation of phosphocholine. Staurosporine, an inhibitor of protein kinases, did not affect the ATP-induced formation of choline. ATP significantly stimulated arachidonic acid release in a dose-dependent manner in the range between 0.01 and 0.5 mmol/L. DL-Propranolol hydrochloride (propranolol), an inhibitor of phosphatidic acid phosphohydrolase, significantly inhibited the ATP-induced release of arachidonic acid. 1,6-Bis(cyclohexyloximinocarbonylamino)-hexane (RHC-80267), a potent and selective inhibitor of diacylglycerol lipase, reduced ATP-induced arachidonic acid release. Quinacrine, a phospholipase A2 inhibitor, suppressed ATP-induced arachidonic acid release. Both propranolol and RHC-80267 markedly inhibited the ATP-induced synthesis of 6-ketoprostaglandin F1 alpha, a stable metabolite of prostacyclin. These results strongly suggest that extracellular ATP activates phosphatidylcholine-hydrolyzing phospholipase D independently of protein kinase C in aortic smooth muscle cells and that the arachidonic acid release induced by extracellular ATP is mediated, at least in part, through phosphatidylcholine hydrolysis by phospholipase D activation.
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PMID:Involvement of phosphatidylcholine hydrolysis by phospholipase D in extracellular ATP-induced arachidonic acid release in aortic smooth muscle cells. 908 84

The effects of the diacylglycerol lipase inhibitor 1,6-bis-(cyclohexyloximinocarbonyl-amino)-hexane (RHC 80267) and the phospholipase A2 inhibitor N-(p-amylcinnamoyl)anthranilic acid (ACA) on insulin secretion and 86Rb+ efflux in mouse pancreatic islets were studied. RHC 80267 (35 microM) and ACA (100 microM) inhibited glucose (16.7 mM)-induced insulin secretion, but did not inhibit insulin secretion induced by K+ (40 mM) or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 0.16 microM). K+ (40 mM) or TPA (0.16 microM) potentiated glucose (16.7 mM)-induced insulin secretion, and prevented inhibition of glucose (16.7 mM)-induced insulin secretion by RHC 80267 and ACA. In comparison, potentiation of glucose-induced insulin secretion by albumin-bound arachidonic acid (AA; 200 microM total; 10 microM free unbound) failed to counteract inhibition of glucose-induced insulin secretion by RHC 80267 or ACA, suggesting that inhibition of insulin secretion by these agents was not mediated by a decrease in AA accumulation in islets. Determination of 86Rb+ efflux, a marker of K+ channel activity, revealed that both RHC 80267 and ACA stimulated K+ efflux from islets. These effects of RHC 80267 and ACA were observed at both 3.3 and 16.7 mM glucose and persisted in Ca2+-free medium, suggesting that they may represent an opening of ATP-sensitive K+ channels. RHC 80267-mediated stimulation of 86Rb+ efflux was not mimicked by the diacylglycerol analog TPA (0.16 microM) and was not prevented by the diacylglycerol kinase inhibitor R 59022 (50 microM), suggesting that stimulation of 86Rb+ efflux did not reflect a conditional increase in diacylglycerol or in phosphatidic acid upon inhibition of diacylglycerol lipase. In contrast, TPA (0.16 microM) attenuated RHC 80267 and ACA stimulation of 86Rb+ efflux. Addition of AA (200 microM total; 10 microM free unbound) stimulated 86Rb+ efflux, suggesting that stimulation of 86Rb+ efflux by RHC 80267 and ACA was not due to a decrease in AA accumulation. This stimulation by AA was not dependent on AA metabolism because it persisted in the presence of the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA; 50 microM) or the cyclooxygenase inhibitor indomethacin (50 microM). In contrast to RHC 80267 and ACA, AA stimulation of 86Rb+ efflux was attenuated in Ca2+-free medium, probably implicating Ca2+-sensitive K+ channels in AA regulation of 86Rb+ efflux. Parallel experiments with diazoxide (100 microM) revealed that RHC 80267 and ACA mimicked the effects of diazoxide, a specific activator of ATP-sensitive K+ channels in islets, on both insulin secretion and 86Rb+ efflux. In conclusion, it is suggested that RHC 80267 and ACA, independently of their action on AA release, may inhibit glucose-induced insulin secretion by the opening of ATP-sensitive K+ channels in islets.
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PMID:Inhibition of glucose-induced insulin secretion by the diacylglycerol lipase inhibitor RHC 80267 and the phospholipase A2 inhibitor ACA through stimulation of K+ permeability without diminution by exogenous arachidonic acid. 917 12

Activation of beta adrenergic receptors in the isolated rabbit heart by catecholamines stimulates prostacyclin (PGI2) synthesis, which is inhibited by adenosine 3'5'-cyclic monophosphate (cAMP). The purpose of this study was to determine if activation of beta adrenergic receptors in cultured coronary endothelial cells (CEC) of rabbit heart with isoproterenol (ISOP) stimulates PGI2 synthesis and if cAMP inhibits the synthesis of this prostanoid and to investigate the underlying mechanism. Incubation of CEC with ISOP increased production of cAMP and PGI2, measured as immunoreactive cAMP and 6-keto-prostaglandin F1alpha, (6-keto-PGF1alpha), respectively. Forskolin, an activator of adenylyl cyclase, increased cAMP accumulation and inhibited ISOP-stimulated 6-keto-PGF1alpha synthesis. 8-(4-chlorophenyl-thio) cAMP also inhibited ISOP-induced 6-keto-PGF1alpha production. However, miconazole, an inhibitor of adenylyl cyclase, reduced cAMP accumulation and enhanced ISOP-stimulated 6-keto-PGF1alpha synthesis in CEC. ISOP-induced 6-keto-PGF1alpha synthesis was attenuated by C2-ceramide, an inhibitor of phospholipase D (PLD) by propranolol, a beta-AR antagonist that also inhibits phosphatidate phosphohydrolase and by the diacylglycerol lipase inhibitor 1,6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC 80267). Acetylcholine (ACh) induced 6-keto-PGF1alpha synthesis was also inhibited by these agents. Both ISOP and ACh increased PLD activity, which was inhibited by C2-ceramide but not by RHC 80267 or propranolol. ACh but not ISOP increased phospholipase A2 activity in CEC. ISOP- but not ACh-induced increase in PLD activity was attenuated by forskolin and 8-(4-chlorophenyl-thio)-adenosine 3'-5'-cyclic monophosphate and augmented by miconazole. These data suggest that beta adrenergic receptors activation promotes PGI2 synthesis in the CEC by selective activation of PLD and that cAMP decreases PGI2 synthesis by decreasing PLD activity. Moreover, beta adrenergic receptors activated PLD appears to be distinct from that stimulated by ACh.
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PMID:Beta adrenergic receptor stimulated prostacyclin synthesis in rabbit coronary endothelial cells is mediated by selective activation of phospholipase D: inhibition by adenosine 3'5'-cyclic monophosphate. 919 Aug 34

CD95 ligand is a cytotoxic cytokine that induces apoptosis. Here we report that CD95-mediated apoptosis of human malignant glioma cells is associated with arachidonic acid (AA) release. Inhibitors of phospholipase A2, phospholipase C or diacylglycerol lipase have minor effects on AA release and fail to modulate apoptosis. Formation of two AA metabolites generated during CD95-dependent apoptosis is attenuated by the lipoxygenase inhibitor, nordihydroguaretic acid (NDGA). NDGA also blocks CD95 ligand-induced apoptosis. This effect is independent of antioxidant properties of NDGA. Lipoxygenase may thus play a critical role in CD95 ligand-induced apoptosis of human malignant glioma cells.
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PMID:Lipoxygenase inhibitors block CD95 ligand-mediated apoptosis of human malignant glioma cells. 919 95

NKR-P1A protein has been implicated in the triggering of NK-mediated natural killing contributing to target cell recognition by NK cells. The aim of the present work was to assess whether NKR-P1A receptor triggering also induced arachidonic acid (AA) generation and to determine the possible role of this event on granule release and cytotoxicity. We demonstrated that activation of fresh peripheral blood rat NK cells by cross-linking with the anti-NKR-P1A 3.2.3 mAb induced calcium-dependent AA release, which is due to the activation of cytosolic phospholipase A2 (cPLA2), secretory phospholipase A2 (sPLA2), and diacylglycerol/monoacylglycerol lipase. We also documented the presence of a type II sPLA2 activity in the supernatant fluids from NKR-P1A-activated rat NK cells, suggesting that AA and lysophospholipids could be mobilized from the outside of the cell. The involvement of AA-generating enzymes in NKR-P1A-induced cytotoxic functions was also investigated. Treatment of effector cells with arachidonyl trifluoromethylketone, a cPLA2 inhibitor; p-bromophenacylbromide, a sPLA2 inhibitor; or RHC80267, a diacylglycerol lipase inhibitor, led to a partial inhibition of the redirected lysis against P815 target cells as well the granule content release induced by NKR-P1A cross-linking. A complete abolishment of these events was observed when the cells were simultaneously incubated with all three inhibitors. Taken together, our results support a crucial role for the arachidonate-generating enzymes in the induction of lytic activity of NK cells directly or by leading to generation of additional mediators that can play a role in the context of NK cell activation and cytotoxic functions.
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PMID:NKR-P1A stimulation of arachidonate-generating enzymes in rat NK cells is associated with granule release and cytotoxic activity. 920 Apr 68

The mechanism(s) of chronic airway inflammation in cystic fibrosis (CF) remains poorly understood. We studied Ca2+-induced release of arachidonic acid (AA), a precursor of proinflammatory lipid mediators, in epithelial cell lines with the deltaF508 mutation in CF transmembrane conductance regulator (CFTR) gene and in those lacking this mutation or cells in which this mutation was corrected by a functional CFTR gene transfer. We found that: (i) the mutant cells manifested an abnormally high Ca2+-induced AA release as compared to controls, (ii) AA release appeared to be catalyzed by a phospholipase A2 (PLA2) but not by phospholipase C followed by diacylglycerol lipase, and (iii) either correction of the CFTR-mutation or inhibition of PLA2 activity rectified this AA release abnormality. Taken together, our results suggest that CFTR mutation is associated with an intrinsic abnormality in AA release by epithelial cells carrying the deltaF508 mutation and suggest that the mechanism of chronic airway inflammation in CF, at least in part, involves this abnormality. These results also partly explain the effectiveness of high-dose ibuprofen therapy in arresting the progression of destructive lung disease in CF. Furthermore, they raise the possibility that correction of abnormal AA release by inhibiting PLA2 activity may improve the therapeutic benefits of ibuprofen.
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PMID:Cystic fibrosis gene mutation (deltaF508) is associated with an intrinsic abnormality in Ca2+-induced arachidonic acid release by epithelial cells. 921 68


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